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Large Scale Plasmid Preps

1. Prepare an o/n culture of 100-200ml in Terrific broth with 75ug/ml ampicillin.

2. Spin down cells 5K, 5 min

3. Resuspend in 5ml of 25 mM Tris, 10 mM EDTA pH8.0 per 200ml

4. Add 10 ml of freshly prepared 1% SDS, 0.2M NaOH (50ml = 1ml 10M NaOH added to 44ml DDW then 5ml 10% SDS). Swirl to lyse cells completely and then add 7.5 ml KoAc solution per 200ml

5. Spin 7 K 10 min

6. Pour supernatant through cheese cloth into SS34 tube and add 11mls of isopropanol (for a 200ml prep).

7. Spin 7 K 10 min. Drain off all supernantant, being careful to wipe around the inside neck of the bottle to remove all the isopropanol.

8. Resuspend pellet in 2.4 ml TE. When dissolved thoroughly, add 4.4g CsCl dissolve 1st (handy hint) then add and 40ul 10mg/ml EtBr. Make sure all CsCl is in solution

9. Layer plasmid solution under 8.5 mls of CsCl/TE p1.47 in a Ti70 tube and spin O/N at 50 K.

CsCl/TE [1.47] =

Dissolve 76.68 gms of CsCl to every 100 mls TE

10. Pull band in 1.0ml or less. Extract 3 times against butanol saturated in TE (first extraction into ethidium waste, remainder into butanol waste).

11. Add 1.0ml DDW, estimate volume and add 2.5 vol. ethanol (no salt). Do not chill. Spin at 3K in benchtop for 10min (provided there was a good band to begin with, otherwise in ultracentriguge for 20' 20K). Pour off s/n and remove residue with a gilson, resuspend in 350ul TE. Read OD 260/280 on 5ul and reprecipitate remainder in ependorf with 35ul 5M NaCl and 1.0ml ethanol. Resuspend at 1ug/l

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This page is maintained by David Bowtell (bowtell@ariel.ucs.unimelb.edu.au) using HTML Author. Last modified on 10/24/95.